Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice
Wenqiang Yin, Kazuo Okamoto, Asuka Terashima, Warunee Pluemsakunthai, Takehito Ono, Taku Ito-Kureha, Shizuo Akira, Yoshinobu Hashizume, Roland Baron, Satoshi Ueha, Kouji Matsushima, Martin M. Matzuk, Yuji Mishina, Hiroshi Takayanagi
Wenqiang Yin, Kazuo Okamoto, Asuka Terashima, Warunee Pluemsakunthai, Takehito Ono, Taku Ito-Kureha, Shizuo Akira, Yoshinobu Hashizume, Roland Baron, Satoshi Ueha, Kouji Matsushima, Martin M. Matzuk, Yuji Mishina, Hiroshi Takayanagi
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Research Article Bone biology Immunology Muscle biology

Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

Abstract

The immune system is not only essential for host defense, but it is also involved in tissue maintenance and disease pathogenesis. Macrophages play a key role in tissue repair, fibrosis, and tumorigenesis, but the mechanisms underlying their multifunctionality have not been fully explored. Here, we identified Mrep (Ly6ChiCX3CR1loPDPN+CD9+) as a crucial subset of macrophages for muscle regeneration after muscle injury. Muscle regeneration required Mrep-derived activin A, which was produced via the TLR4/TIR domain–containing adapter-inducing interferon-β/TANK-binding kinase 1/interferon regulatory factor 3/7 signaling pathway in response to muscle injury. Mrep exerted pathological effects by secreting activin A in a model of genetically induced heterotopic ossification (HO), which was suppressed by TLR4 inhibition. Thus, this study elucidates the context-dependent functions of macrophages and the link between injury and HO, suggesting that Mrep is a potential therapeutic target for regenerating muscles and suppressing HO.

Authors

Wenqiang Yin, Kazuo Okamoto, Asuka Terashima, Warunee Pluemsakunthai, Takehito Ono, Taku Ito-Kureha, Shizuo Akira, Yoshinobu Hashizume, Roland Baron, Satoshi Ueha, Kouji Matsushima, Martin M. Matzuk, Yuji Mishina, Hiroshi Takayanagi

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Figure 3

Ly6ChiCX3CR1loPDPN+CD9+ MDMs preferentially express activin A in injured muscle.

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Ly6ChiCX3CR1loPDPN+CD9+ MDMs preferentially express activin A in injured...
(A) UMAP visualization of cells isolated from muscle at 1 dpi. Phylogenetic tree showing cluster hierarchy and cell numbers (in parentheses). (B) Heatmap showing the top 10 DEGs by log2FC (adjusted P < 0.001) across clusters. Typical cell surface molecules were selected for naming. Expression of Ly6C is shown in Supplemental Figure 5C. (C) UMAP showing the expression of Inhba in each cluster. (D) Dot plot showing the relative Inhba expression across 3 subclusters of MDMs. (E and F) RT-qPCR results showing relative Inhba mRNA expression in muscles at 1 dpi from mice treated with 150 μL vehicle (n = 7) or clodronate (n = 9) (E) and from mice treated with 0.25 mg isotype IgG (n = 8) or 0.25 mg anti-Ly6G antibodies (n = 7) (F). (G) Gating strategy for sorting 4 subpopulations of Ly6ChiCX3CR1lo MDMs based on the expression of PDPN and CD9. (H) Flow cytometric analysis confirming the purity of sorted cells. (I) RT-qPCR results showing relative Inhba expression in indicated subpopulations sorted from muscles on 1 dpi (n = 4 for each). The expression in the PDPN–CD9– fraction was set to 1. (J) ELISA results showing the concentration of activin A in the culture supernatants of indicated MDMs sorted from muscles on 1 dpi. Supernatants were collected 48 hours after seeding. (K) Flow cytometric histogram showing IL-7R expression in indicated MDMs at 1 dpi. The P values were calculated using unpaired 2-tailed t test (D, E, and J) or 1-way ANOVA with Tukey’s multiple-comparison test (I). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM, and symbols represent individual mice (D, E, I, and J).